J Acupunct Meridian Stud 2016;9(1):16e21

Available online at www.sciencedirect.com

Journal of Acupuncture and Meridian Studies

journal homepage: www.jams-kpi.com

RESEARCH ARTICLE

Preemptive Analgesia with Acupuncture

Monitored by c-Fos Expression in Rats
André T.A. Gonçalves de Freitas 1,*, Lino Lemonica 2,
Julio De Faveri 3, Sergio Pereira 4, Maria D. Bedoya Henao 5

1
Department of Morphology, Universidade Estadual Paulista Júlio de Mesquita Filho,
UNESP-Botucatu, São Paulo, Brazil
2
Department of Anesthesiology, Universidade Estadual Paulista Júlio de Mesquita Filho,
UNESP-Botucatu, São Paulo, Brazil
3
Department of Pathology, Universidade Estadual Paulista Júlio de Mesquita Filho,
UNESP-Botucatu, São Paulo, Brazil
4
Department of Biological Sciences, Universidade Estadual Paulista Júlio de Mesquita Filho,
UNESP-Botucatu, São Paulo, Brazil
5
Clinic of Acupuncture and Homeopathy, Universidade Estadual Paulista Júlio de Mesquita
Filho, UNESP-Botucatu, São Paulo, Brazil

Available online 1 September 2015

Received: Mar 18, 2015 Abstract

Revised: Jul 28, 2015 Pain behavior and awareness are characterized by heightened alertness and anxiety,
Accepted: Aug 5, 2015 which begin to disappear as soon as the curative process starts. The present study aimed
to quantify c-fos expression in rat spinal cords and brains after a surgical stimulus and
KEYWORDS with preoperative or postoperative acupuncture. Animals were randomly divided into
acupuncture; preoperative and postoperative groups and were then further divided into control,
analgesia; manual acupuncture (MA), or electroacupuncture (EA) groups. Expression of c-fos was
c-fos protein; quantified using immunohistochemistry. The collected data were analyzed using the t
pain test at a 5% probability level. Presurgery and postsurgery spinal cord c-fos expressions
were similar in all of the treatment groups. In the control rats, c-fos expression was high-
er before surgery than after surgery, contradicting the expected outcome of acupuncture
and preemptive analgesia. After treatment, the expression of c-fos in the brains of the

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://
creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any
medium, provided the original work is properly cited.
* Corresponding author. Department of Morphology, Bioscience Institute UNESPdSão Paulo State University, 18618-970 Botucatu,
São Paulo, Brazil.
E-mail: andretagf@gmail.com (A.T.A. Gonçalves de Freitas).

pISSN 2005-2901 eISSN 2093-8152

http://dx.doi.org/10.1016/j.jams.2015.08.002
Copyright ª 2016, Medical Association of Pharmacopuncture Institute.
Preemptive Analgesia with Acupuncture
rats in the MA and the EA groups was reduced compared with that of the rats in the con-
trol group. These findings suggest that acupuncture used as preemptive analgesia in rats
is a useful model for studying its application in human treatment.
1. Introduction
Pain is an uncomfortable sensorial and emotional experi-
ence associated with an actual or psychogenic lesion, and
an important mediator of response to harmful agents. Pain
control can involve a multimodal approach that includes
pharmacological and nonpharmacological techniques, pro-
moting relaxation and reduction of the painful stimulus [1].
Among the nonpharmacological techniques with recognized
analgesic effects are manual acupuncture (MA) and
electroacupuncture (EA), which activate the sensitive-
discriminative system to stimulate a suppressive pain
response [2,3]. Pain control can be started prophylactically,
and the preemptive administration of opioids or local an-
esthetics can reduce postoperative pain, hyperalgesia, and
morphine intake [4,5]. This suggests that an analgesic
intervention before surgery will produce a better outcome
than the same intervention after surgery [6].
Pain response may influence the cellular genome, causing
important changes in gene transcription and protein syn-
thesis. Genes that show a rapid change in expression after
neuronal activity are known as early or immediate response
genes. The structural events that occur in the nervous sys-
tem after a pain stimulus are related to the activation of
these genes, which include proto-oncogenes such as c-fos
and c-jun. The identification of c-fos expression in the spinal
cord and brain is a reliable method for assessing the efficacy
of analgesic treatments [7].
The repetition of a stimulus is capable of reducing c-fos
expression [8,9]. Mapping of the brain areas associated with
the analgesic effects of acupuncture by means of c-fos
expression in either anaesthetized or restrained animals
has helped to elucidate its mechanisms of action [7].
This study aimed to quantify c-fos expression in the
spinal cord and brain of rats after a painful stimulus in order
Figure 1 Manual acupuncture (MA) and electroacupuncture (EA) in rats. (A) MA in E36. (B) EA in E36. (C) EA in B67 (Ting).
17
to evaluate the preoperative and postoperative effects of
acupuncture.
2. Materials and methods
2.1. Animal treatments
Thirty-six adult male Wistar rats, from the Animal Depart-
ment of Universidade Federal de São Paulo (UNIFESP),
Brazil, weighing 210e235 g were maintained five to each
cage in order to avoid stress caused by overcrowding, under
normal temperature and controlled light, and with food and
water provided ad libitum. All experimental protocols were
approved by the Animal Care and Use Committee of UNI-
FESP and were in accordance with National Institutes of
Health guidelines on animal care.
Animals were distributed randomly to control, preoper-
ative, and postoperative groups, with 12 animals in each
group, and then subdivided into MA and EA groups, with six
animals in each group. All animals were anesthetized with
45 mg/kg sodium thiopental until no motor sensibility could
be observed. The preoperative groups were submitted to
MA or EA at 100 Hz frequency in the following points:
Stomach 36, Kidney 1, and Bladder 67, bilaterally (Fig. 1).
The needles were removed after 20 minutes and surgery
was started. An approximately 1-cm long incision was made
in the plantar region of the left paw using a scalpel blade.
After this, superficial and deep tissues were dissected.
After 60 minutes, animals were sacrificed and perfused. In
the postoperative groups, animals were stimulated for 20
minutes, at the same points after surgery, and then sacri-
ficed and perfused.
Rats were euthanized by injecting 75 mg/kg sodium
thiopental intraperitoneally, and perfusion was started 60
18
minutes later if the animals did not manifest any signs of
consciousness. To remove the spinal cord and brain, ani-
mals were placed in a horizontal ventral decubitus position
above a wooden platform.
2.2. Immunohistochemistry
The c-fos immunoreactive (Fos-ir) cells were detected
using a conventional avidinebiotineimmunoperoxidase
technique to localize an antiserum raised against a syn-
thetic N-terminal fragment of human Fos protein (Ab-5;
Oncogene Science, Cambrige, MA, USA) [10]. Briefly, free-
floating sections were pretreated with hydrogen peroxi-
dase, followed by sodium borohydride. Sections were then
treated with normal goat serum (1:100) and 0.3% Triton X-
100 for 2 hours and incubated with the primary antiserum at
a dilution of 1:3,000 in potassium phosphate buffer at room
temperature for 24 hours. Subsequently, the sections were
incubated with a secondary antibody (goat anti-rabbit IgG,
1:200; Vector Laboratories, Inc., Burlingame, CA, USA) for
90 minutes at room temperature and treated with the
avidinebiotin complex (1:100; Vector) for a further 90 mi-
nutes. Staining was completed using the nickel-intensified
diaminobenzidine reaction. Between steps, the sections
were rinsed in 0.002 M potassium phosphate buffer (pH
7.4). The tissue was agitated on a rotator between each
incubation and rinse step. Sections were mounted on
gelatin-coated slides, dried, dehydrated, and covered with
a coverslip [7].
2.3. Counting c-fos-positive nuclei
As described in previous studies [11], the nomenclature and
nuclear boundaries defined in Swanson’s Stereotaxic Rat
Brain Atlas were used in this study [12]. C-fos-immunoreac-
tive nuclear profiles in different areas of the brain were
counted using a BX50 Olympus microscope (Olympus America
Inc., Melville, NY, USA) coupled to a Macintosh-based image
Figure 2 Photomicrograph illustrating stress-induced c-fos-immunoreactive cells. (AeC) In the spinal cord. (DeF) In the brain.
Original magnifications 40.
A.T.A. Gonçalves de Freitas et al.
analysis system (Apple Computer Inc., Cupertino, CA, USA)
and Neurozoom software (The Scripps Research Institute/
Mount Sinai School of Medicine, La Jolla, CA, USA). The
boundaries of the brain areas were identified using adjoining
Nissl-stained sections. A template or outline was constructed
for each brain nucleus or subnucleus based on the shape and
size of the region. The location and relative size of each
template is shown in Fig. 2. The c-fos-positive nuclei within
each area were counted bilaterally (where possible) in two
consecutive sections/animal and the mean value is reported
as the number of Fos-ir cells/10,000 mm2. This counting
procedure allowed a reliable, time-effective analysis of c-
fos expression in 41 brain areas [11].
For spinal cord c-fos-positive nuclei counting, the dorsal
horn was analyzed. It was previously shown [13] that
physiological stimulation of rat primary sensory neurons
causes increased Fos-like immunoreactivity in the nuclei of
postsynaptic neurons of the spinal dorsal horn.
2.4. Statistical analyses
The average, median, and standard deviation of each group
(control, preoperative and postoperative) were calculated.
To verify the effect of surgery and of each treatment
(control, MA, and EA), a variance analysis was conducted,
calculating the F statistics and p values for comparisons
among the three treatments in pre- and postoperative
conditions, and t and p values for pre- and postoperative
comparisons. The results were considered statistically sig-
nificant when p < 0.05.
3. Results
3.1. Spinal cord
Results from the statistical analysis of Fos-ir cell counts in
the spinal cord and brain are shown in Table 1, and
Preemptive Analgesia with Acupuncture 19

Table 1 The average number of c-fos-immunoreactive cells/104 mm2 ( standard deviation) in the spinal cord and brain.
Control Pre MA Pre EA Post MA Post EA
Spinal cord 9.36  5.51 8.12  4.62 14.33  11.75 12.23  8.11 13.38  15.84
Brain 94.31  13.9b,* 47.94  12.32a 42.98  5.85a 46.26  4.45a 39.49  8.49a
* Different lowercase letters (a and b) indicate statistically significant differences between experimental groups.
EA Z electroacupuncture; MA Z manual acupuncture.

immunohistochemistry photomicrographs illustrating the 3.2. Brain

effect of different treatments on c-fos expression are shown
in Fig. 3. There were no significant differences in c-fos
expression after treatment, and likewise in the pre- and
posttreatment groups, neither MA nor EA caused a significant
change in c-fos positivity. There was also no difference in c-
fos expression when MA or EA were compared during the pre-
and postsurgical period.
Table 2 summarizes the results of a comparative analysis of
c-fos expression between control animals and those treated
with MA or EA. Significant differences between all treat-
ment groups compared with the control group were
observed. Comparisons between the same treatments in
the pre- or postsurgical periods revealed no significant

Figure 3 Diagrams illustrating the templates and relative sizes of the different brain areas in which c-fos-immunoreactive cells
were counted [11]. The levels were based on Swanson’s Stereotaxic Atlas of the Brain [12]. Abbreviations list: ACA Z anterior
cingulate area; ARH Z arcuate nucleus of the hypothalamus; BLA Z basolateral nucleus of the amygdala; BST Z bed nucleus of the
stria terminalis; CA1 Z field CA1; CA2 Z field CA2; CA3 Z field CA3; CeA Z central nucleus of the amygdala; CM Z central medial
nucleus of the thalamus; CoA Z cortical nucleus of the amygdala; DG Z dentate gyrus; DMH Z dorsomedial nucleus of the hy-
pothalamus; DR Z dorsal raphe nucleus; ENT Z entorhinal area; LC Z locus coeruleus; LH Z lateral habenula; LHA Z lateral
hypothalamic area; LSd Z lateral septal nucleus, dorsal part; LSi Z lateral septal nucleus, intermediate part; LSv Z lateral septal
nucleus, ventral part; MeA Z medial nucleus of the amygdala; Mop Z primary motor area; MPT Z medial pretectal nucleus;
MPO Z medial preoptic area; NTS Z nucleus tractus solitarius; PBl Z parabrachial nucleus, lateral part; PH Z posterior hypo-
thalamus; PIR Z piriform area; PVT Z paraventricular nucleus of the thalamus; PVa Z anterior periventricular nucleus of the
hypothalamus; PVp Z posterior periventricular nucleus of the hypothalamus; PVHmpd Z paraventricular nucleus of the hypo-
thalamus, medial parvicellular part, dorsal zone; RM Z nucleus raphe magna; RO Z nucleus raphe obscurus; RPA Z nucleus raphe
pallidus. SCH Z suprachiasmatic nucleus; SS Z somatosensory area; SUMI Z supramammillary nucleus; VISC Z visceral area.
20
Table 2 Comparative analysis between control
and treatment groups with respect to the number of c-fos-
immunoreactive cells in the brain.
Comparison Statistical difference p The pre- and postoperative acupuncture treatments
Control vs. pre-MA 46.369 < 0.001
Control vs. pre-EA 51.327 < 0.001
Control vs. post-MA 48.047 < 0.001
Control vs. post-EA 54.813 < 0.001
EA Z electroacupuncture; MA Z manual acupuncture.
differences in c-fos expression. The difference in
the average number of c-fos-immunoreactive cells/104 mm2
( standard deviation) between pre- and postsurgical MA
groups was 1.678 (p > 0.05), and between pre- and post-
surgical EA groups it was 3.486 (p > 0.05). Likewise, there
was no significant difference in c-fos positivity between
different treatments in the same period (4.958 for preop-
erative MA and EA, and 6.766 for postoperative MA and EA;
p > 0.050).
4. Discussion
4.1. Spinal cord
The absence of any significant difference (p > 0.100) in c-
fos expression between pre- and postoperative treatments
indicates that animals exposed to pre- and postoperative
stimuli experienced a similar level of stress, and conse-
quently, had similar c-fos expression levels in the dorsal
horn of the spinal cord. Regardless of the treatment used,
none of the animals were completely sedated, to the point
where c-fos expression would be repressed. Although the
differences in c-fos expression were not significant, the
highest expression was in animals treated with preemptive
EA, suggesting that this technique causes more stress than
the other treatments. It is also noteworthy that the “Ting”
points selected in this study (Bladder 67 and Kidney 1) are
localized in the edges of the toes, which are more sensitive
to pain stimuli due to intense innervations. It was previ-
ously reported [3] that 2 Hz electrical stimulation at
traditional acupuncture sites induced the release of ence-
phalin in the spinal cord of rats, providing a more effective
analgesia than we observed in this study.
The absence of significant differences in c-fos expression
between treatments in postoperative animals might be
explained by individual variations, particularly in the ani-
mals treated with MA and EA, and may not necessarily be
associated with the treatment effect per se. Other studies
have shown conflicting results when using opioids to control
conditional pain, probably as a result of methodological
differences [14].
Against our expectations, MA in pre- and postoperative
conditions did not cause a significant reduction in c-fos
expression. The E36 point has been shown to have an
analgesic and sedative effect (7), and is thus expected to
reduce c-fos expression. This might indicate that the ani-
mals were subjected to too much stress in these experi-
ments. Previous studies have shown that B67 stimulation,
but not stimulation of less important visual function
A.T.A. Gonçalves de Freitas et al.
acupoints, resulted in a significant increase in the number
of c-fos-positive cells in the primary visual cortex [15].
However, we observed no significant differences in c-fos
positivity after B67 stimulation.
failed to have any analgesic effect, probably because no
endogenous opioids were released. There was, therefore,
an increase in c-fos expression, mainly because the initial
stimuli of analgesia by MA or EA can be painful. Analgesia by
acupuncture relies on the manipulation of a physiological
process, and thus may not lead to a complete loss of
sensation or pain.
4.2. Brain
Changes in c-fos expression in the brain indicated that
preemptive analgesia with either AM or EA reduced stress.
Treated animals showed a significant reduction of c-fos
expression, corroborating previous findings [3,16,17]. The
comparison between control animals and those in the
different treatment groups (Table 2) showed a very signif-
icant difference in c-fos expression (p < 0.001) for each
treatment, proving that they are effective compared with
the control. We observed that, regardless of the treatment,
MA and EA were able to induce analgesia and increase the
pain threshold of the animals, possibly with increased
opioid and monoamine release.
With respect to the number of Fos-ir cells, our study
revealed that there was no statistical difference between
pre- and postoperative groups (p > 0.050) after either MA
or EA treatment. This indicates that animals subjected to
painful stimuli pre- and postoperatively suffered a similar
level of stress and manifested a similar c-fos expression
level in the brain.
When comparing groups treated at the same time (pre-
or postoperatively) but with different treatments (MA or
EA), there were indications that EA reduced pain (although
the differences were not significant), in agreement with
previous studies. Recent studies [18] have shown that EA is
effective in the control of pain and symptoms for which MA
has always been ineffective. Moreover, an increased num-
ber of Fos-ir neurons in the hippocampal region following
EA was recently demonstrated [19]. Despite there being no
significant differences in spinal cord c-fos expression, the
brain results corroborate studies in which acupuncture
needle penetration produced only a low level of stress
[20e22], suggesting that it could be used as an alternative
to, or in combination with, pharmacological intervention.
Our study provides strong evidence that acupuncture
can induce analgesia by reducing c-fos expression. Using
acupuncture to provide preemptive analgesia in rats rep-
resents an important model for studying the physiological
effects and mechanisms of this technique, and the results
of these studies could be applicable to future human
treatment.
Disclosure statement
The authors declare that they have no conflicts of interest
and no financial interests related to the material of this
manuscript.
Preemptive Analgesia with Acupuncture 21

References [12] Swanson LW. Brain Maps: Structure of the Rat Brain.
Amsterdam, The Netherlands: Elsevier; 1992.
[13] Hunt SP, Pini A, Evan G. Induction of c-fos-like protein in
[1] Chou R, Huffman LH. Nonpharmacologic therapies for acute
spinal cord neurons following sensory stimulation. Nature.
and chronic low back pain: a review of the evidence for an
1987;328:632e634.
American Pain Society/American College of Physicians clinical
[14] Suzan E, Midbari A, Treister R, Haddad M, Pud D, Eisenberg E.
practice guideline. Ann Intern Med. 2007;147:492e504.
Oxycodone alters temporal summation but not conditioned
[2] Chang FC, Tsai HY, Yu MC, Yi PL, Lin JG. The central seroto-
pain modulation: preclinical findings and possible relations to
nergic system mediates the analgesic effect of electro-
mechanisms of opioid analgesia. Pain. 2013;154:1413e1418.
acupuncture on ZUSANLI (ST36) acupoints. J Biomed Sci. 2004;
[15] Lee H, Park HJ, Kim SA, Lee HJ, Kim MJ, Kim CJ, et al.
11:179e185.
Acupuncture stimulation of the vision-related acupoint (Bl-67)
[3] Chen X, Geller E, Adler M. Electrical stimulation at traditional
increases c-Fos expression in the visual cortex of binocularly
acupuncture sites in periphery produces brain opioid-
deprived rat pups. Am J Chin Med. 2002;30:379e385.
receptor-mediated antinociception in rats. J Pharmacol Exp
[16] Guo HF, Tian J, Wang X, Fang Y, Hou Y, Han J. Brain substrates
Ther. 1996;277:654e660.
activated by electroacupuncture (EA) of different frequencies
[4] Katz J, Melzack R. Pain ‘memories’ in phantom limbs: review
(II): role of Fos/Jun proteins in EA-induced transcription of
and clinical observations. Pain. 1990;43:319e336.
preproenkephalin and preprodynorphin genes. Brain Res Mol
[5] Machida M, Imamura Y, Usui T, Asai T. Effects of preemptive
Brain Res. 1996;43:167e173.
analgesia using continuous subcutaneous morphine for post-
[17] Guo HF, Tian J, Wang X, Fang Y, Hou Y, Han J. Brain substrates
operative pain in scoliosis surgery: a randomized study.
activated by electroacupuncture of different frequencies (I):
J Pediatr Orthop. 2004;24:576e580.
comparative study on the expression of oncogene c-fos and
[6] McQuay HJ. Pre-emptive analgesia. Br J Anaesth. 1992;69:
genes coding for three opioid peptides. Brain Res Mol Brain
1e3.
Res. 1996;43:157e166.
[7] de Medeiros M, Canteras N, Suchecki D, Mello L. Analgesia and
[18] Inoue M, Nakajima M, Hojo T, Kitakoji H, Itoi M. Spinal nerve
c-Fos expression in the periaqueductal gray induced by elec-
root electroacupuncture for symptomatic treatment of lumbar
troacupuncture at the Zusanli point in rats. Brain Research.
spinal canal stenosis unresponsive to standard acupuncture: a
2003;973:196e204.
prospective case series. Acupunct Med. 2012;30:103e108.
[8] Papa M, Pellicano MP, Welzl H, Sadile AG. Distributed changes
[19] Wang LF, Xu CL, Xiong KR. Effects of electroacupuncture on
in c-Fos and c-Jun immunoreactivity in the rat brain associ-
expression of tyrosine hydroxylase and c-fos in hippocampal
ated with arousal and habituation to novelty. Brain Res Bull.
CA 1 area in ketamine-addiction rats. Zhen Ci Yan Jiu. 2011;
1993;32:509e515.
36:23e27.
[9] Melia KR, Ryabinin AE, Schroeder R, Bloom FE, Wilson MC.
[20] Kaptchuk TJ, Kelley JM, Conboy LA, Davis RB, Kerr CE,
Induction and habituation of immediate early gene expression
Jacobson EE, et al. Components of placebo effect: rando-
in rat brain by acute and repeated restraint stress. J Neurosci.
mised controlled trial in patients with irritable bowel syn-
1994;14:5929e5938.
drome. BMJ. 2008;336:999e1003.
[10] Ryabinin AE, Wang YM, Finn DA. Different levels of Fos
[21] Vickers AJ, Maschino AC. The Acupuncture Trialists’ Collabo-
immunoreactivity after repeated handling and injection stress
ration: individual patient data meta-analysis of chronic pain
in two inbred strains of mice. Pharmacol Biochem Behav.
trials. Acupunct Med. 2009;27:126e127.
1999;63:143e151.
[22] Vickers AJ, Cronin AM, Maschino AC, Lewith G, MacPherson H,
[11] Medeiros MA, Canteras NS, Suchecki D, Mello LE. c-Fos
Foster NE, et al. Acupuncture for chronic pain: individual
expression induced by electroacupuncture at the Zusanli point
patient data meta-analysis. Arch Intern Med. 2012;172:
in rats submitted to repeated immobilization. Braz J Med Biol
1444e1453.
Res. 2003;36:1673e1684.